This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. With the advent of high mass accuracy/resolution mass analyzers suitable for the analysis of covalently modified peptides and proteins, chemical crosslinking has flourished as a technique for mapping interacting domains and providing low resolution structures of protein complexes. Crosslinking complements other structural methods such as crystallography or electron microscopy because it can study structure in solution under a variety of biochemical conditions, and is suited to examining dynamic complexes. However, existing crosslinking reagents are not suitable for the analysis of large, multi-protein complexes because of their proclivity to hydrolyze in aqueous buffers. This leads to low yields of crosslinked peptides and a corresponding high yield of "dead-end" modified peptides, in which hydrolysis has occurred on one half of the reagent. The goal of this project is to extend the utility and scale of chemical crosslinking, which has generally been limited to the study of simple, binary protein interactions, to larger ensembles of cellular machinery that catalyze many fundamental cellular processes such as the fusion of synaptic vesicles or mRNA splicing. This project encompasses: 1) the design and synthesis of new crosslinking reagents, 2) the development of methodology to enrich specifically crosslinked peptides and 3) the development of bioinformatic tools to assist in identifying crosslinked MS/MS spectra resulting from a large pool of interacting proteins. The new methodology applies the chemistry of reductive amination to increase crosslinking yields and allow selective enrichment of true crosslinked peptides from unmodified and dead-end modified peptides.